The P2Y2 Receptor Control of Hyperglycemia-induced Insulin Resistance in Human Hepatocytes: A Unified Mechanism for Metformin

Abstract

According to the International Diabetes Federation, in 2021, 537 million adults were living with diabetes, and another 541 million adults were at increased risk of developing T2D (Facts & Figures, 2021.) Obesity, the most significant contributing risk fact for T2D, is also the foundation of non-communicable diseases (NCDs), which combined, are the most considerable threat humanity faces – 63% of all deaths may be attributed to cardiovascular disease, cancer, chronic respiratory disease, and diabetes (Noncommunicable Diseases, 2022). Macroeconomic simulations suggest that the financial burden of NCDs over the next two decades is a cumulative output loss of $47 trillion (The Global Economic Burden of Non-Communicable Diseases, 2011). Therefore, the most fruitful intervention mechanism to positively influence these projections is addressing the problem at its core – obesity, diabetes, and metabolic syndrome.

Deleting the ectonucleotidase CD39 leads to liver insulin resistance in mice; however, it remains unknown whether this phenotype is due to a lack of extracellular adenosine generation or excessive nucleotides accumulation and signaling. We hypothesized that the collection of extracellular ATP signaling through the P2Y2 receptor (P2Y2R) mediates insulin resistance in human hepatocytes. After assessing the mRNA for the eight known P2Y receptors, only the P2Y2, P2Y6, P2Y11, and P2Y12 mRNAs were detected in cultured HepG2 cells, a human hepatocytes model. Stimulation of the cells with P2Y6- and P2Y11-selective agonists failed to induce intracellular Ca2+ mobilization. In contrast, ATP and UTP induced significant Ca2+ signaling dose-dependently, which was abolished by pretreatment of the cells with ARC-118925XX, a selective and competitive P2Y2R antagonist suggesting that the P2Y2R is the only functional Gq-coupled P2Y receptor expressed in HepG2 cells. In addition, we found that stimulation of P2Y2R induced MAPK signaling, including phosphorylated ERK1/2, JNK, and p38, but noticeably suppressed the AKT pathway. Interestingly, stimulation of the cells with ATP or UTP dose-dependently blocked insulin-induced AKT phosphorylation and signaling but potentiated ERK1/2 signaling, indicating a selective disruption of the insulin-AKT signaling axis by P2Y2R.

Furthermore, we found a notable secretion of ATP into the extracellular space when exposing HepG2 cells to high glucose concentrations (25mM) compared with normal glucose (5.5 mM), independent of osmolarity. We also found that extracellular nucleotides signaling through P2Y2R induced a dose-dependent reduction in hepatocyte glucose uptake and stimulated the two primary mechanisms of hepatic glucose production – gluconeogenesis and glycogenolysis. Thus, we conclude that P2Y2R is a mediator of sustaining hepatic glucose production and insulin resistance during hyperglycemia, possibly through activating pro-inflammatory signaling pathways and subsequent inhibition of insulin signaling. We also present evidence that sequestration of phosphatidylinositol 4,5-bisphosphate (PIP2) away from the insulin receptor-PI3K pathway offers a novel insulin resistance mode.

Metformin is the first line of pharmacologic therapy to treat type 2 diabetes mellitus (T2DM). The mechanism of action has been heavily debated since the 1950s when French scientists deemed it "Glucophage" or glucose eater. However, metformin's antidiabetic effects are related to increasing insulin sensitivity, decreasing hepatic glucose production, and decreasing glucose uptake from the digestive tract. Pharmacokinetic studies indicate circulating concentrations of metformin are between 10 and 40μM, while the broad range of effects observed in prior studies occurred at supratherapeutic concentrations exceeding 1mM.

This study demonstrates that clinically relevant metformin concentrations and other antidiabetic biguanides block glucose-stimulated ATP secretion (GSAS). Additionally, lysosome alkalizing agents produce similar inhibitory effects on GSAS. From the evidence, metformin likely induces the alkalization of the lysosomal compartment and reduces ATP cargo and/or release probability - the suspected target of metformin at therapeutic concentrations is Aspartate 231 (NHE9) conserved in Na+/H+ exchangers 7/9 (NHE7/9).